The present application relates to a bilirubin oxidase mutant having thermal stability. More specifically, the present application relates to a bilirubin oxidase mutant having prescribed levels or more of heat resistance in addition to enzymatic activity and its contribution to the durability of a biocathode in an enzymatic fuel cell.
An “enzyme” is a biocatalyst for allowing many reactions relative to the maintenance of life to smoothly proceed under a mild condition in vivo. This enzyme turns over in vivo, is produced in vivo depending on the situation and exhibits its catalytic function.
At present, technologies for utilizing this enzyme in vitro have already been put into practical use or studied towards practical implementation. For example, a technology for utilizing an enzyme has been developed in various technical fields such as the production of a useful substance, the production, measurement or analysis of energy-related substance, the environmental preservation and the medical treatment. In relatively recent years, technologies regarding an enzyme cell which is one kind of a fuel cell (see, for example, JP-A-2004-71559), an enzyme electrode, an enzyme sensor (a sensor for measuring a chemical substance utilizing an enzymatic reaction) and the like have also been proposed.
Since a chemical main body of this enzyme is a protein, the enzyme has properties that it is denatured by the degree of heat or pH. For that reason, enzymes have low stability in vitro as compared with other chemical catalysts such as metal catalysts. Accordingly, when an enzyme is utilized in vitro, it is important to allow the enzyme to work more stably in response to an environmental change and to maintain an activity thereof.
When an enzyme is utilized in vitro, approaches such as a method for artificially modifying the nature or function of the enzyme itself and a method for devising the environment of a site where the enzyme works are employed. With respect to the former method, it is generally carried out that the base sequence of a gene encoding a protein is artificially modified, the thus modified gene is expressed in an organism such as Escherichia coli to produce an artificially mutated protein, and the protein mutant having functions and natures adapted to the use purpose is then subjected to separation (screening) (see, for example, JP-A-2004-298185).
The “bilirubin oxidase” as referred to herein is an enzyme which catalyzes a reaction for oxidizing bilirubin into biliverdin and is one kind of enzyme belonging to a multicopper oxidase (a general term of an enzymes having plural copper ions in the active center). This enzyme has hitherto been widely used as an inspection reagent of liver function and the like (a measurement reagent of bilirubin in a blood serum) in the clinical laboratory examination. In recent years, this enzyme is also regarded as a catalyst for realizing an electrochemical four-electron reduction reaction of oxygen on a cathode side of the foregoing enzyme cell.
Under circumstances where expectations for utilizing this bilirubin oxidase in vitro are rising, a technology for investigating the same enzyme having more excellent thermal stability (see, for example, JP-A-2006-68003) and a technology for stably maintaining the enzymatic activity of the same enzyme over a longer period of time (see, for example, JP-A-2000-83661) have also been proposed.
In consideration of the utilization of a bilirubin oxidase in vitro, it is necessary that the thermal stability is more enhanced. However, this bilirubin oxidase involves a problem that the enzymatic activity is reduced to not more than 20% by heating at 60° C. for one hour. For example, in the field of an enzyme cell, since the bilirubin oxidase has the lowest thermal stability among a group of enzymes to be utilized and is remarkably low in the thermal stability as compared with enzymes on an anode side (for example, glucose dehydrogenase and diaphorase), it is not suitable to put an enzyme cell into practical use. Also, though there is a choice to substitute this bilirubin oxidase with laccase which is a multicopper oxidase, this laccase involves not only a problem regarding the heat resistance but a problem that the enzymatic activity at room temperature in a neutral pH region is remarkably low as compared with the bilirubin oxidase.
The durability of an enzymatic biofuel cell is generally determined by the stability of the immobilized enzyme on its electrode. There has been a focus on the extended active enzyme lifetimes at anode surfaces but limited studies on the stability of the cathode enzyme.
Bilirubin oxidase (BO, EC 1.3.3.5), which belongs to the sub-family of multicopper oxidase (MCO), has been widely employed as the cathode enzyme of an enzymatic biofuel cell, because this enzyme has higher activity than the other MCOs at neutral pH (4,6,7). Studies have shown that an enzymatic biofuel cell with BO reached the maximum power density of 5.0 mW/cm2 at 0.5 V by the introduction of some new technologies (Sakai, H. et. al., Energy Environ. Sci. 2009, 2, 133; Sakai et. al., ECS Trans., 2009, 16(38), 9) and these studies demonstrated that a biofuel cell has power density enough to apply in practical use. The thermo-stability of BO, however, is much lower than that of the anode enzymes, and this is problematic for practical use as a biofuel cell.